Leptin, the protein product of the ob gene, is an important circulating signal for regulating body weight, food intake, and energy metabolism in mammals (Zhang Y et al., Nature 372: 425-432, 1994). These actions are elicited through the binding of leptin to a high affinity receptor in the hypothalamus (Tartaglia L A et al., Cell 83: 1263-1271, 1995). Although leptin was initially described as a satiety factor that regulates the size of adipose tissue, leptin has many other diverse biological functions. These functions are elicited by the binding of leptin to receptor proteins that are expressed in numerous tissues.
Leptin has been shown to correct the sterility defect in leptin-deficient mice and accelerate puberty when administered to normal mice (Chehab et al. Nature Genet. 12:318-320, 1996). Humans defective in either leptin or the leptin receptor are sterile and sexually immature, supporting leptin""s role in reproduction (Stroebel et al., Nature Genet. 18: 213-215, 1998; Clement et al., Nature 392: 398-401, 1998). Other roles for leptin include a regulator of hematopoeisis (Cioffi et al., Nature Medicine 2: 585-589, 1996; Gainsford et al., Proc. Natl. Acad. Sci. USA 93: 14564-14568, 1996), angiogenesis (Bouloumie et al., Circ. Res. 83: 1059-1066, 1998; Sierra-Honigmann et al., Science 281: 1683-1686, 1998), glucose metabolism (Kamohara et al., Nature 389: 374-377, 1997), and proinflammatory immune responses (Loffreda et al., FASEB J 12:57-65, 1998; Lord et al., Nature 394: 897-901, 1998). Since malnutrition is the leading cause of diminished immunity and increased susceptibility to infection, leptin therapy may also augment the immune response in compromised individuals (Flier, Nature Medicine 4: 1124-1125, 1998).
Although first thought to be produced exclusively by the adipocyte (fat cell), it is now known that leptin is produced in the placenta (Hassink et al., Pediatrics 100:e1-e6, 1997; Masuzaki et al., Nature Med. 3: 1029-1033, 1997), gastric epithelium (Bado et al., Nature 394:790-793, 1998), and the mammary gland, as more fully described below (Smith-Kirwin et al., J. Clin. Endocrinol. Metab. 83: 1810-1813, 1998, herein incorporated by reference). The function of placental leptin seems to be as a regulator of fetal growth. Prematurity accounts for a large proportion of infant morbidity, primarily due to respiratory distress, immaturity of organ systems, and poor nutrition. As more fully described below, the premature infant is leptin-deficient, due to early separation from the placenta at a time when they have inadequate adipose tissue. A method to administer leptin to premature and/or poorly growing infants is highly desirable.
Previous leptin therapies rely on the use of recombinant leptin. The administration of recombinant leptin is performed intravenously, intramuscularly, intraperitoneal, and through other parenteral routes to treat obesity, diabetes, and reproductive abnormalities. The amount of leptin that can be delivered by these means is limited by recombinant leptin""s poor solubility and by local reactions in skin that occur in response to high doses (Friedman and Halaas, Nature 395: 763-770, 1998). In rodent models, it has been shown that the ability to optimize the means of administration of leptin may greatly influence its therapeutic effectiveness. Leptin has been shown to be more potent when administered as a subcutaneous infusion than when administered by daily intraperitoneal injections (Halaas et al., Proc. Natl. Acad. Sci. USA 94: 8878-8883, 1997). Direct administration of leptin into the cerebrospinal fluid (Halaas et al., Proc. Natl. Acad. Sci. USA 94: 8878-8883, 1997) and by gene therapy means (Chen et al., Proc. Natl. Acad. Sci. USA 93: 14795-14799, 1996; Murphy et al., Proc. Natl. Acad. Sci. USA 94: 13921-13926, 1997) have also been shown to be effective for weight loss in rodents, but for safety and ethical concerns have not been tried in humans.
Previous methods involving leptin therapies rely on the use of recombinant leptin. Purification of recombinant leptin involves denaturation of the protein and subsequent renaturation steps. The renatured leptin must then be solubilized, which is problematic because the recombinant leptin aggregates at high concentrations and much protein loss occurs (Guisez et al., Protein Expression Purification 12: 249-258, 1998). The renatured leptin must then be tested for bioactivity.
Therefore, it is desirable to provide a method for administering leptin that does not require the use or purification of recombinant leptin that can be administered orally, intraveneously, subcutaneously, intramuscularly, intraperitoneally or by other parenteral means. As stated above, a method to administer leptin to premature and/or poorly growing infants is highly desirable.
As more fully described below, the present invention overcomes the problems associated with previous forms of leptin therapy and includes a novel method and composition for administering native leptin that can be administered to subjects who have deficient leptin levels or require leptin therapy to treat a disorder.
The present invention includes a method and composition of native leptin in milk fat globules. This composition may be administered orally, intravenously, intramuscularly, intraperitoneally, or by other enteral or other parenteral means. In one embodiment, the invention includes a method for treating growth-related problems in newborns.
The invention includes a composition for the administration of leptin comprising isolated milk fat globules wherein the milk fat globules contain a native form of leptin.
The invention further includes isolated milk fat globules that are derived from a milk-producing mammal that produces milk fat globules containing a native form of leptin. The mammal may be selected from the group consisting of humans, cows, rats, mice, and goats.
The invention includes a medium that is effective for suspending the milk fat globules and is safe to administer to humans. Preferably the medium is selected from the group consisting of physiologic saline, water, intravenous fluid, milk, human breast milk, baby formula, and any other medium that is safe for human or infant administration.
The invention includes suspending the isolated milk fat globules in the medium at a concentration between about 50 ng/ml leptin and about 100 ng/ml leptin. Preferably, the isolated milk fat globules are suspended at a concentration of about 75 ng/ml.
Further, the invention includes isolated milk fat globules where at least a portion of the leptin is contained within the milk fat globules.
The invention includes a method for administering leptin that includes administering to a subject an effective amount of isolated native leptin-containing milk fat globules where the isolated native leptin-containing milk fat globules have been suspended in a medium to produce suspended milk fat globules.
The invention includes subjects that have a condition selected from the group consisting of obesity, sterility, sexual immaturity, malnutrition, compromised immunity, psychiatric disorders, diabetes, and subjects with a condition that can be treated by the administration of leptin.
Still further, the invention includes a subject selected from a group consisting of a premature infant, a full-term infant having low leptin levels, a low birth weight infant, and an infant with a condition that can be treated with leptin.
The present invention further includes administering an effective amount of suspended milk fat globules by an administration method selected from the group consisting of oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraperitoneal administration, and other enteral or parenteral routes of administration.
Still further, the present invention includes a method for administering leptin to infants that includes administering to the infant an effective amount of isolated native leptin-containing milk fat globules wherein the isolated native leptin-containing milk fat globules are suspended in a medium to produce suspended milk fat globules.
The invention includes a medium that is effective for suspending the milk fat globules and is safe to administer to infants. Preferably, the medium is selected from the group consisting of physiologic saline, water, intravenous fluid, milk, human breast milk, baby formula, soy milk, and any other medium that is safe for infant administration.
The invention includes administering an effective amount of leptin to an infant. Preferably, the effective amount is about 0.5 ng to about 1.0 ng of leptin per gram of infant body weight per feeding.